U.S. patent application number 12/783818 was filed with the patent office on 2011-11-24 for high yield dialysis-free process for producing organosoluble regenerated silk fibroin.
This patent application is currently assigned to TAIPEI MEDICAL UNIVERSITY. Invention is credited to Chien Chung CHEN, Yun-Ting HSU, Dian-Yu JI, Sheng-Yang LEE, Hong-Da WU, Jen-Chang YANG.
Application Number | 20110288273 12/783818 |
Document ID | / |
Family ID | 44973014 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110288273 |
Kind Code |
A1 |
YANG; Jen-Chang ; et
al. |
November 24, 2011 |
HIGH YIELD DIALYSIS-FREE PROCESS FOR PRODUCING ORGANOSOLUBLE
REGENERATED SILK FIBROIN
Abstract
The invention relates to a simple and high yield process for
producing a regenerated silk fibroin which does not need dialysis.
Particularly, a process of the invention is characterized in that
the silk fibroin is precipitated by applying a shear stress and/or
changing the solvent power of the fibroin solution. The process of
the invention simplifies the process of producing silk fibroin and
greatly shortens the process time to 1 to 2 hours, whereas the
conventional dialysis process is complex and needs around 2 to 3
days. In addition to reducing the time needed, the process of the
invention can increase productivity of silk fibroin by at least
8%.
Inventors: |
YANG; Jen-Chang; (Taipei
City, TW) ; LEE; Sheng-Yang; (Taipei City, TW)
; CHEN; Chien Chung; (Taipei City, TW) ; HSU;
Yun-Ting; (Taipei City, TW) ; WU; Hong-Da;
(Taipei, TW) ; JI; Dian-Yu; (Taipei, TW) |
Assignee: |
TAIPEI MEDICAL UNIVERSITY
Taipei City
TW
|
Family ID: |
44973014 |
Appl. No.: |
12/783818 |
Filed: |
May 20, 2010 |
Current U.S.
Class: |
530/353 |
Current CPC
Class: |
C07K 14/43586
20130101 |
Class at
Publication: |
530/353 |
International
Class: |
C07K 1/30 20060101
C07K001/30 |
Claims
1. A process of producing a regenerated silk fibroin, comprising
the steps of: (a) dissolving degummed silk fibroins in a salt
containing solution to give a fibroin solution; and (b) applying a
shear stress to the fibroin solution for a time sufficient to make
the silk fibroin precipitate, thereby producing a regenerated silk
fibroin.
2. The process of claim 1, wherein in step (b), the shear stress is
provided by stirring, homogenizer or high speed homogenizer.
3. The process of claim 2, wherein in step (b), the stirring is at
3,000 to 20,000 rpm to provide the stress tress.
4. The process of claim 2, wherein in step (b), the stirring is at
3,000 to 10,000 rpm to provide the shear stress.
5. The process of claim 2, wherein in step (b), the stirring is at
5,000 to 10,000 rpm to provide the shear stress.
6. The process of claim 2, wherein in step (b), the stirring is at
5,000 to 8,000 rpm to provide the shear stress.
7. The process of claim 1, wherein the salt containing solution in
step (a) comprises an aqueous cupri-ethylenediamine solution, an
aqueous ammonia solution of cupric hydroxide (Schweitzer's
reagent), an aqueous alkaline solution of cupric hydroxide and
glycerol (Roe's reagent), an aqueous lithium bromide solution, an
aqueous solution of the chloride, nitrate or thiocyanate of
calcium, magnesium or zinc, or an aqueous sodium thiocyanate
solution.
8. The process of claim 7, wherein the salt containing solution is
an aqueous solution of the chloride or nitrate of calcium or
magnesium.
9. The process of claim 7, wherein the salt containing solution is
an aqueous solution of CaCl.sub.2.
10. The process of claim 7, wherein the salt containing solution is
a solution of CaCl.sub.2/C.sub.2H.sub.5OH/H.sub.2O.
11. The process of claim 1, further comprising a degumming step of
silk fibroin before step (a).
12. A process of producing a regenerated silk fibroin, comprising
the steps of: (a) dissolving degummed silk fibroins in a salt
containing solution to give a fibroin solution; (b') changing the
solvent power of the fibroin solution; and (c) applying a shear
stress to the fibroin solution to the fibroin solution for a time
sufficient to make the silk fibroin precipitate, thereby producing
a regenerated silk fibroin.
13. The process of claim 12, wherein in step (b'), the solvent
power of the fibroin solution is changed by changing the proportion
of the salts in the fibroin solution.
14. The process of claim 13, wherein the solvent power of the
fibroin solution is changed by lowering the proportion of salts in
the solution.
15. The process of claim 12, wherein in step (b'), the solvent
power of the fibroin solution is changed by increasing the
proportion of water in the solution.
16. The process of claim 12, wherein the salt containing solution
of step (a) comprises an alcohol.
17. The process of claim 12, wherein the salt containing solution
of step (a) comprises an alcohol and in step (b'), the solvent
power of the fibroin solution is changed by increasing the
proportion of alcohol in the solution.
18. The process of claim 12, further comprising a degumming step of
silk fibroin before step (a).
19. The process of claim 12, wherein the salt containing solution
of step (a) comprises an aqueous cupri-ethylenediamine solution, an
aqueous ammonia solution of cupric hydroxide (Schweitzer's
reagent), an aqueous alkaline solution of cupric hydroxide and
glycerol (Roe's reagent), an aqueous lithium bromide solution, an
aqueous solution of the chloride, nitrate or thiocyanate of
calcium, magnesium or zinc, or an aqueous sodium thiocyanate
solution.
20. The process of claim 19, wherein the salt containing solution
of step (a) is an aqueous solution of the chloride or nitrate of
calcium or magnesium.
21. The process of claim 19, wherein the salt containing solution
of step (a) is an aqueous solution of CaCl.sub.2.
22. The process of claim 19, wherein the salt containing solution
of step (a) is a solution of
CaCl.sub.2/C.sub.2H.sub.5OH/H.sub.2O.
23. The process of claim 14, wherein the proportion of salts of the
solution is lowered by adding multivalent acid ions to the fibroin
solution.
24. The process of claim 23, wherein the multivalent acid ion is
selected from the group consisting of SO.sub.4.sup.2-,
C.sub.2O.sub.4.sup.2-, CO.sub.3.sup.2-, and PO.sub.4.sup.3-.
25. The process of claim 23, wherein the multivalent acid ion is
SO.sub.4.sup.2- or PO.sub.4.sup.3-.
26. The process of claim 23, wherein the multivalent acid ion is
derived from the salt containing Na.sup.+ and/or K.sup.+.
27. The process of claim 19, wherein the salt containing solution
in step (a) is a solution of CaCl.sub.2 or Ca(SCN).sub.2.
28. The process of claim 23, wherein the multivalent acid ion is
derived from a salt selected from the group consisting of
K.sub.2SO.sub.4, KHSO.sub.4, Na.sub.2SO.sub.4, NaHSO.sub.4,
Na.sub.3PO.sub.4, Na.sub.2HPO.sub.4, NaH.sub.2PO.sub.4,
K.sub.3PO.sub.4, K.sub.2HPO.sub.4, and KH.sub.2PO.sub.4.
29. The process of claim 15, wherein the solution contains more
than 90 wt % of water.
30. The process of claim 15, wherein the solution contains 0.1 to 3
wt %, 0.1 to 2 wt %, 0.1 to 1 wt % or 0.1 to 0.99 wt % of
fibroin.
31. The process of claim 30, wherein the solution contains 0.1 to
0.99 wt % of fibroin.
32. The process of claim 30, wherein the solution contains 0.1 to
0.95 wt %, 0.5 to 0.95 wt %, 0.5 to 0.92 wt % or 0.6 to 0.9 wt % of
fibroin.
Description
FIELD OF THE INVENTION
[0001] The invention provides a simple and high yield process for
producing a regenerated silk fibroin which does not need dialysis.
Particularly, the process of the invention is characterized in that
the silk fibroin is precipitated by applying a shear stress to
it.
BACKGROUND OF THE INVENTION
[0002] Silk protein is regarded as a natural biomaterial, and
developments of new uses of silk threads in various fields other
than that of clothing are underway. Silk fibroin has very good
properties such as high strength and flexibility, biocompatibility,
blood compatibility, water permeability and oxygen permeability, so
it can be used in surgery as implant material and in tissue
engineering applications. In addition, silk fibroin can be used as
a cell culture matrix, as a substratum for cultivation of cells, as
a burn wound dressing membrane, as an enzyme-immobilization
material, and in an oral dosage form.
[0003] Silk is mainly composed of fibroin and sericin, and the
fibroin has 19% hydrophilic side chains containing a heavy chain
with a molecular weight of 325 kD (45% glycine, 30% alanine, 12%
serine) and a light chain with a molecular weight of 25 kD (15%
asparate, 11% glycine, 14% alanine, 11% serine). In addition, the
sericin in silk fibers is in an amount of around 25% by weight and
it has 76% hydrophilic side chains. The sericin will cause
inflammation, so the silk fibers should be degummed with hot water
containing surfactants before medical applications. Fibrous
products based on silk fibroins have excellent mechanical
properties. Nevertheless, silk fibroin has Tg ranging from
170-175.degree. C. and the temperature of transiting random-coil to
.beta.-structure conformation is 212.degree. C. Therefore, when the
temperature rises to 280.degree. C., the silk fibroin will start to
cleave. Thus, silk fibroin is usually processed by solution process
rather than by melt process.
[0004] To conduct the solution process, silk fibroin need to be
dissolved in a salts containing aqueous solution first. U.S. Pat.
No. 5,252,285 indicates that fibroin is known to be soluble in
certain high ionic strength aqueous salt solutions, for example,
aqueous lithium thiocyanate (LiSCN), sodium thiocyanate (NaSCN),
calcium thiocyanate (Ca(SCN).sub.2), magnesium thiocyanate
(Mg(SCN).sub.2), calcium chloride (CaCl.sub.2), lithium chloride
(LiCl), lithium bromide (LiBr), zinc chloride (ZnCl.sub.2),
magnesium chloride (MgCl.sub.2), copper salts such as copper
nitrate (Cu(NO.sub.2).sub.2), copper ethylene diamine
(Cu(NH.sub.2CH.sub.2CH.sub.2NH.sub.2).sub.2 (OH).sub.2) and
Cu(NH.sub.3).sub.4(OH).sub.2 and Ajisawa's reagent
(CaCl.sub.2/ethanol/water). The processing of silk fibroin solution
is difficult due to salt concentration increasing when solvent
evaporates at an elevated temperature. Even after the salts removed
by dialysis out of such aqueous salt/fibroin solutions, the
concentration of this fibroin solution is usually too dilute to
spin fiber threads. More commonly, the organosoluble silk fibroin
is first harvested by freeze drying process from the dialyzed
solution. Then, fibers can be spun from fibroin solution dope that
was prepared by dissolved silk fibroin solution in an organic
solvent. US 2007/0187862 A1 provides for concentrated aqueous silk
fibroin solutions and an all-aqueous mode for preparation of
concentrated aqueous fibroin solutions by dialysis that avoids the
use of organic solvents, direct additives, or harsh chemicals. This
application indicates that dialysis of the solution against a
hygroscopic polymer is also sufficient to control water content in
the formation of silk hydrogels.
[0005] Concerning the bottleneck of silk fibroin mass-production,
the dialysis process is usually time-consuming and difficult to
scale up. Moreover, during the dialysis procedure, the
intermolecular hydrogen bonds of silk fibroins gradually form, so
molecules of silk fibroins tend to form crystals where the second
structure of silk fibroin gradually becomes silk crystal form I
(organic solvent soluble) and silk crystal form II (organic solvent
insoluble) from random-coil conformation. Furthermore, gelation of
silk fibroin renders its second structure unstable, so the
solubility of regenerated silk fibroin is hard to maintain.
[0006] Sung-Won Ha et al. dissolves silk fibroin with the calcium
nitrate tetrahydrate-methanol system and uses wet spinning method
to spin regenerated fibroin fiber (Biomacromolecules, 2003, 4 (3),
pp 488-496). A solvent system is developed to use a solution
containing 1-butyl-3-methylimidazolium chloride as solvent, which
can dissolve silk fibroin without dialysis process (Phillips, D.
M.; Drummy, L. F.; Conrady, D. G.; Fox, D. M.; Naik, R. R.; Stone,
M. O.; Trulove, P. C.; De Long, H. C.; Mantz, R. A. Journal of the
American Chemical Society, 2004, 126, 14350-14351). However, this
solvent system is expensive and is not appropriate for industrial
production. U.S. Pat. No. 7,285,637 applies a formic acid solution
containing a small amount of salts to break the disulfide bonds
between heavy (350 kDa) and light (27 kDa) chains of silk fibroin
so that the dissolution of the silk fibroin in the solution can be
facilitated. Nevertheless, there is a serious molecular chain
cleavage of silk fibroin in the solvent system. Furthermore, it was
reported that silk fibroin can dissolve in hexafluoroisopropanol
solvent after 5 months (Zarkoob, S.; Reneker, D. H.; Ertley, D.; R.
K. Eby; Hudson, S. D. Synthetically spun silk nanofibers and a
process for marking the same, 6110590, 2000). The long dissolving
time makes the method impractical for mass production.
[0007] Therefore, there is still a need to develop a more
convenient and efficient process for producing a silk fibroin.
SUMMARY OF THE INVENTION
[0008] The invention provides a process of producing a regenerated
silk fibroin, comprising the steps of: [0009] (a) dissolving
degummed silk fibroins in a salt containing solution to give a
fibroin solution; and [0010] (b) applying a shear stress to the
fibroin solution for a time sufficient to make the silk fibroin
precipitate, thereby producing a regenerated silk fibroin.
[0011] The invention further provides a process of producing a
regenerated silk fibroin, comprising the steps of: [0012] (a)
dissolving degummed silk fibroins in a salt containing solution to
give a fibroin solution; [0013] (b') changing the solvent power of
the fibroin solution; and [0014] (c) applying a shear stress to the
fibroin solution to the fibroin solution for a time sufficient to
make the silk fibroin precipitate, thereby producing a regenerated
silk fibroin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the XRD results of the calcium sulfate
dihydrate (CaSO.sub.4.2H.sub.2O), the fibroin along with the
calcium sulfate dihydrate (CaSO.sub.4.2H.sub.2O) produced by the
process according to the invention, and the potassium sulfate.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The invention relates to a simple and high yield process for
producing a dialysis-free regenerated silk fibroin by changing
phase equilibrium. The phase equilibrium can be changed by, for
example, applying a shear stress or changing solvent power. Various
means of changing phase equilibrium can be used in combination to
increase the precipitation rate. The process of the invention
simplifies the process of producing silk fibroin and greatly
shortens the processing time to 1 to 2 hours, whereas the dialysis
process is complex and needs around 2 to 3 days. In addition to
reducing the time needed, the process of the invention can increase
productivity of silk fibroin by at least 8%.
[0017] In one aspect, the invention provides a process of producing
a regenerated silk fibroin, comprising the steps of: [0018] (a)
dissolving degummed silk fibroins in a salt containing solution to
give a fibroin solution; and [0019] (b) applying a shear stress to
the fibroin solution for a time sufficient to make the silk fibroin
precipitate, thereby producing a regenerated silk fibroin.
[0020] In another aspect, in addition to the application of shear
stress, the process can further comprise a step of changing the
solvent power to increase the precipitation rate. The dissolution
of silk fibroin is correlated with the power of the solvent in the
solution. Silk fibroin dissolves more easily when the power of the
solvent is weak, and vice versa. The power of the solvent in the
solution, in turn, is correlated with the proportion of the
components in the solvent. Therefore, the power of the solvent can
be lowered by lowering the proportion of salts in the solution,
increasing the proportion of water in the solution, or increasing
the proportion of the alcohol in the solution if alcohol is
included.
[0021] The term "fibroin" includes selected proteins; preferably,
fibroins are obtained from a solution containing degummed silkworm
silk proteins. The silkworm silk proteins are obtained, for
example, from Bombyx mori. The silk proteins which are used to
obtain the degummed silk fibroins in the process of this invention
can be cocoons, raw silk, waste cocoons, raw silk waste, bisu
(un-peelable cocoons), silk fabric waste, bourette, and the like.
Alternatively, the silk proteins suitable for use in the present
invention can be obtained from a solution containing a genetically
engineered silk, such as from bacteria, yeast, mammalian cells,
transgenic animals or transgenic plants. According to the
invention, the term "degummed silk fibroin" refers to the silk
fibroin obtained by degumming silk protein.
[0022] The term "regenerated silk fibroin" refers to the silk
fibroin generated through the process of the invention.
[0023] The term "shear stress" refers to an external force acting
on an object or surface parallel to the slope or plane in which it
lies.
[0024] Before step (a) of the process according to the invention,
the initial degumming of silk protein is done by using a treatment
based on a NaHCO.sub.3 solution or other degumming methods known in
the art and described in the scientific literature and then the
degummed silk fibroin is obtained. The term "degumming" means the
partial or complete removal of sericin. The silk protein is
degummed or freed from sericin by any conventional procedure. For
example, it is washed in warm water containing a surface-active
agent or an enzyme according to the need, and then dried.
[0025] In the dissolving step of (a) of the process of the
invention, the degummed silk fibroins are dissolved in a salt
containing solution.
[0026] The salt containing solution which is used in the process of
this invention can comprise an aqueous cupri-ethylenediamine
solution, an aqueous ammonia solution of cupric hydroxide
(Schweitzer's reagent), an aqueous alkaline solution of cupric
hydroxide and glycerol (Roe's reagent), an aqueous lithium bromide
solution, an aqueous solution of the'chloride, nitrate or
thiocyanate of calcium, magnesium or zinc, and an aqueous sodium
thiocyanate solution. It is preferable to use an aqueous solution
of the chloride or nitrate of calcium or magnesium, because of its
low cost and convenience for use. More preferably, an aqueous
solution of CaCl.sub.2 is used. It is more preferable to use a
solution of CaCl.sub.2/C.sub.2H.sub.5OH/H.sub.2O that has high ion
strength (preferably in a mole ratio of 1:2:8). The concentrations
of these aqueous solutions may vary according to the type of
solvent used, the temperature, and the like. Where an aqueous
solution of a metal salt is used, its concentration is generally 5
to 80% by weight, preferably 20 to 70% by weight, and most
preferably 25 to 60% by weight.
[0027] According to the invention, the dissolution of the degummed
silk fibroin in a salt containing solution can be carried out at
higher temperatures, for example higher than 60.degree. C., for 2
to 8 hours with agitation. Preferably, the dissolution is performed
at a temperature of about 70.degree. C. for 4 to 6 hours.
[0028] In step (b) of the process of the invention, a shear stress
is applied to the fibroin solution to induce a phase separation so
that the silk fibroin is regenerated and precipitates from the
solution. According to the invention, the shear stress can be
provided by stirring, homogenizer or high speed homogenizer.
Preferably, 3,000 to 20,000 rpm of shear stress is provided; more
preferably, 3,000 to 10,000, 5,000 to 10,000 or 5,000 to 8,000 rpm
is provided.
[0029] In another aspect, the invention provides a process of
producing a regenerated silk fibroin, comprising the steps of:
[0030] (a) dissolving degummed silk fibroins in a salt containing
solution to give a fibroin solution; [0031] (b') changing the
solvent power of the fibroin solution; and [0032] (c) applying a
shear stress to the fibroin solution for a time sufficient to make
the silk fibroin precipitate, thereby producing a regenerated silk
fibroin.
[0033] In one aspect, in step (b') of the process of the invention,
the solvent power of the solution can be changed by changing the
proportion of the salts in the fibroin solution. In one embodiment,
the solvent power is changed by lowering the proportion of salts in
the solution. One or more multivalent acid ions can be added to the
fibroin solution of step (a) to lower the proportion of salts in
the solution by complexing the metal ion of the salt in the
solution and precipitating. The multivalent acid ion is selected on
the basis of the ability of the multivalent acid ion to combine
with the metal ion of the salt the solution of step (a). That is,
the choice of the multivalent acid ion depends on the metal ions
originally contained in the fibroin solution. Preferably, the acid
ion is selected from the group consisting of SO.sub.4.sup.2-,
C.sub.2O.sub.4.sup.2-, CO.sub.3.sup.2-, and PO.sub.4.sup.3-, and
can be derived from a salt containing Na.sup.+ and/or K.sup.+. For
example, the salt to give the multivalent acid ion can be
K.sub.2SO.sub.4, KHSO.sub.4, Na.sub.2SO.sub.4, NaHSO.sub.4,
Na.sub.3PO.sub.4, Na.sub.2HPO.sub.4, NaH.sub.2PO.sub.4,
K.sub.3PO.sub.4, K.sub.2HPO.sub.4 or KH.sub.2PO.sub.4. Preferably,
the multivalent acid salt is K.sub.2SO.sub.4. The concentration of
the multivalent acid ion should be about equivalent to the
concentration of the metal ions contained in the salt of the
solution of step (a). By addition of the multivalent acid ions, the
concentration of metal ions in the fibroin solution can be
decreased, and the power of the solvent is lowered.
[0034] In one embodiment of the invention, the salt of step (a) is
CaCl.sub.2 or Ca(SCN).sub.2 and the multivalent acid salt to give
the multivalent acid ion is K.sub.2SO.sub.4, KHSO.sub.4,
Na.sub.2SO.sub.4, NaHSO.sub.4, Na.sub.3PO.sub.4, Na.sub.2HPO.sub.4,
NaH.sub.2PO.sub.4, K.sub.3PO.sub.4, K.sub.2HPO.sub.4 or
KH.sub.2PO.sub.4.
[0035] In one aspect, in step (b') of the process of the invention,
the solvent power of the solution can be changed by increasing the
proportion of water in the solution, for example, by adding
de-ionized water to the resulting fibroin solution. Preferably, the
proportion of water in the solution is more than 90 wt %, or the
solution contains less than 5 wt % of fibroin. More preferably, the
solution contains less than 4 wt %, 3 wt %, 2 wt % or 1 wt % of
fibroin, such as 0.1 to 3 wt %, 0.1 to 2 wt %, 0.1 to 1 wt %, 0.1
to 0.99 wt % of fibroin. More preferably, the ranges of fibroin are
0.1 to 0.95 wt %, 0.5 to 0.95 wt %, 0.5 to 0.92 wt % and 0.6 to 0.9
wt %.
[0036] In one aspect, if an alcohol is included in the solution of
the process of the invention as one of the solvents, the solvent
power of the solution can be changed by increasing the proportion
of alcohol in the solution. The alcohol can be, for example,
ethanol.
[0037] After step (c) of the process of the invention, the
re-generated and precipitated silk fibroin is collected and
obtained. According to the invention, the harvesting of the
precipitated silk fibroin can be performed by filtration. In
addition, an centrifugation process can be performed optionally
following the filtration in order to precipitate the silk
fibroin
[0038] After the harvesting step, the precipitated silk fibroin can
be further washed or rinsed by deionized water to remove remaining
salts and then dried to obtain regenerated silk fibroin.
Preferably, the drying is freeze-drying.
[0039] The regenerated silk fibroin obtained from the process of
the invention is organosoluble. The process of the invention is
simple, rapid, and appropriate for mass production, and has high
yield. The regenerated silk fibroin can be used in a variety of
medical applications such as a drug (e.g, small molecule, protein,
or nucleic acid) delivery device, including controlled release
systems, wound closure systems, including vascular wound repair
devices, hemostatic dressings, patches and glues, sutures, and in
tissue engineering applications, such as, for example, scaffolds
for tissue regeneration, ligament prosthetic devices and in
products for long-term or bio-degradable implantation into the
human body. The step of adding multivalent acid ion containing salt
to the fibroin solution to change the solvent power of the fibroin
solution can recover the metal ions used to dissolve degummed and
dried silk fibroins. Particularly, when the solution containing
CaCl.sub.2 or Ca(SCN).sub.2 is used to dissolve degummed and dried
silk fibroins, the multivalent acid ion containing salt,
K.sub.2SO.sub.4, KHSO.sub.4, Na.sub.2SO.sub.4, NaHSO.sub.4,
Na.sub.3PO.sub.4, Na.sub.2HPO.sub.4, NaH.sub.2PO.sub.4,
K.sub.3PO.sub.4, K.sub.2HPO.sub.4 or KH.sub.2PO.sub.4 can be used.
The acid ion of the multivalent acid salt will combine with the
metal ion in the solution and precipitate. For example, CaSO.sub.4
or Ca.sub.3(PO.sub.4).sub.2 can be formed and precipitated.
Subsequently, CaSO.sub.4 or Ca.sub.3(PO.sub.4).sub.2 can be used
together with silk fibroin as bone substitute composites without
removing them.
[0040] The present invention is further defined in the following
examples. It should be understood that the examples, while
indicating preferred embodiments of the inventions, are given by
way of illustration only. From the above discussion and the
examples, one skilled in the art can ascertain the essential
characteristics of this invention, and can make various changes and
modifications of the invention to adapt it to various uses and
conditions without departing from the spirit and scope thereof.
EXAMPLES
Example 1
Preparation of Fibroin Solution
[0041] After the silkworm chrysalis was removed from the cocoon,
the cocoon (30 g) was cleaned (with de-ionized water) and placed
into an autoclave with de-ionized water (3,000 ml) to perform
degumming. Degumming was performed at 121.degree. C. and under
pressure of 1.2 kg/cm.sup.2 for 1 hour. The degummed fibroin was
then cleaned with de-ionized water and dried at 50.degree. C. for
one day to yield the fibroin (20.4.+-.0.4 g). The resulting fibroin
was dissolved in CaCl.sub.2/C.sub.2H.sub.5OH/H.sub.2O (mole
ratio=1:2:8) to yield a solution containing 10 weight % fibroin.
Stirring may be performed at 70.degree. C. for 4 to 6 hours to
fully dissolve the fibroin. The solution was then filtered through
a mesh screen and spun by high speed centrifuge (5,000 rpm,
25.degree. C.) for 20 min twice to remove the impurities.
Example 2
Preparation of Silk Fibroin by Adding Water and Applying Shear
Stress
[0042] The resulting solution was then diluted (with de-ionized
water) to yield a solution containing less than 1 weight % fibroin.
Shear stress (by 5,000-8,000 rpm spin) and heat were then applied
to the resulting solution to generate the fibroin precipitate. The
fibroin was collected and cleaned to remove the salts. After being
cool dried (-48.degree. C.) for one day, fibroin powder was
obtained. The yield was 87.7.+-.3.5%. A comparative example using
the fibroin solution of Example 1 but without applying shear stress
to the solution shows a yield of 70.8.+-.4.7%. Table 1 below shows
the results of different ratios of the silk fibroin
(SF)/(CaCl.sub.2/C.sub.2H.sub.5OH/H.sub.2O)/diluted de-ionized
water (DDW) system according to the process described above.
TABLE-US-00001 TABLE 1 Static State (10 min) SF/solution Diluted +
system/DDW Rate Shear-Induced (Ratio) (w/w) Precipitation (5 min)
0.91/8.18/90.91 1:10 .smallcircle. 0.63/5.63/93.75 1:15
.smallcircle. 0.48/4.29/95.24 1:20 .smallcircle. 0.38/3.46/96.15
1:25 .smallcircle. 0.32/2.90/96.77 1:30 .smallcircle.
0.24/2.20/97.56 1:40 .smallcircle. 0.20/1.76/98.04 1:50
.smallcircle. .smallcircle.: silk fibroin precipitated.
[0043] It can be learned from Table 1 that the silk fibroin can be
precipitated from the solution by adding de-ionized water and by
applying shear stress to the solution.
[0044] The molecular weight of silk fibroin can be indexed by
inherent viscosity measurement. The regenerated SF powder was
dissolved in formic acid to prepare 1 w/v % sample solution. The
viscometer (Cannon Penske Routine Viscometers, Size No. 75) was
filled with 10 mL sample solution at 25.degree. C. for constant
temperature. An experiment was conducted to measure the efflux time
necessary for the sample solution to flow from point b to d. The
value of inherent viscosity, .eta..sub.inh, was calculated by the
following equation:
.eta. r = .eta. .eta. 0 = .rho. t .rho. 0 t 0 ##EQU00001## .eta.
inh = ln .eta. r C = 2.303 log .eta. r C ##EQU00001.2##
where .eta..sub.0 is the viscosity of the solvent, .eta. is the
viscosity of the sample solution, .rho..sub.0 is the density of the
solvent, .rho. is the density of the sample solution, t.sub.0 is
the efflux time of the solvent, t is the efflux time of the sample
solution, and C is the gram of polymer dissolved in 100 ml
solvent.
[0045] The average efflux time to generate the fibroin by the
process according to the present invention is 594.08 seconds
(inherent viscosity: 1.22 dL/g) while the average efflux time to
generate the fibroin by the conventional dialysis process is 625.3
seconds (inherent viscosity: 1.27 dL/g). In view of the inherent
viscosity obtained by the above experiment (corresponding to
molecular weight of the fibroin), the molecular weight of the
fibroin produced by the process of the invention does not have a
significant difference with that of a conventional dialysis
process.
Example 3
Preparation of Silk Fibroin by Adding Multivalent Acid Salt and
Applying Shear Stress
[0046] A solution of fibroin was prepared as described in Example 1
and yielded a solution containing 10 weight % of fibroin. The
solution was diluted with de-ionized water (weight ratio 1:1). A
K.sub.2SO.sub.4 solution was added to the diluted solution
(CaCl.sub.2:K.sub.2SO.sub.4=1:1 (mole)). After stirring to provide
shear stress, the fibroin was precipitated. After being cleaned and
dried, the fibroin and the calcium sulfate dihydrate powders
(CaSO.sub.4.2H.sub.2O) were obtained. The calcium sulfate powders
can be further used together with silk fibroin as bone substitute
composites.
[0047] FIG. 1 shows the XRD results of the calcium sulfate
dihydrate (CaSO.sub.4.2H.sub.2O), the fibroin along with the
calcium sulfate dihydrate (CaSO.sub.4.2H.sub.2O) produced by the
process according to the invention, and the potassium sulfate. The
FIGURE confirms that the precipitate on the surface of the fibroin
is calcium sulfate dihydrate (CaSO.sub.4.2H.sub.2O).
* * * * *